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Cancer-associated cachexia (CAC) is a wasting syndrome drastically reducing efficacy of chemotherapy and life expectancy of patients. CAC affects up to 80% of cancer patients, yet the mechanisms underlying the disease are not well understood and no approved disease-specific medication exists. As a multiorgan disorder, CAC can only be studied on an organismal level. To cover the diverse aetiologies of CAC, researchers rely on the availability of a multifaceted pool of cancer models with varying degrees of cachexia symptoms. So far, no tumour model syngeneic to C57BL/6 mice exists that allows direct comparison between cachexigenic- and non-cachexigenic tumours.MCA207 and CHX207 fibrosarcoma cells were intramuscularly implanted into male or female, 10-11-week-old C57BL/6J mice. Tumour tissues were subjected to magnetic resonance imaging, immunohistochemical-, and transcriptomic analysis. Mice were analysed for tumour growth, body weight and -composition, food- and water intake, locomotor activity, OCHX207, but not MCA207-tumour-bearing mice exhibited major clinical features of CAC, including systemic inflammation, increased plasma IL-6 concentrations (190 pg/mL, P ≤ 0.0001), increased energy expenditure (+28%, P ≤ 0.01), adipose tissue loss (-47%, P ≤ 0.0001), skeletal muscle wasting (-18%, P ≤ 0.001), and body weight reduction (-13%, P ≤ 0.01) 13 days after cancer cell inoculation. Adipose tissue loss resulted from reduced lipid uptake and -synthesis combined with increased lipolysis but was not associated with elevated beta-adrenergic signalling or adipose tissue browning. Muscle atrophy was evident by reduced myofibre cross sectional area (-21.8%, P ≤ 0.001), increased catabolic- and reduced anabolic signalling. Deletion of IL-6 from CHX207 cancer cells completely protected CHX207In this study, we present CHX207 fibrosarcoma cells as a novel tool to investigate the mediators and metabolic consequences of CAC in C57BL/6 mice in comparison to non-cachectic MCA207-tumour-bearing mice. IL-6 represents an essential trigger for CAC development in CHX207-tumour-bearing mice.

Background: Atherosclerosis, the leading cause of cardiovascular disease responsible for the majority of deaths worldwide, cannot be sufficiently explained by established risk factors including hypercholesterolemia. Elevated plasma homocysteine is an independent risk factor for atherosclerosis and is strongly linked to cardiovascular mortality. However, the role of homocysteine in atherosclerosis is still insufficiently understood. Previous research in this area has been also hampered by the lack of reproducible in-vivo models of atherosclerosis that resemble the human situation.Methods: Aortic specimens obtained from balloon injured rabbits fed either diet deficient in B vitamins, supplemented with 1% cholesterol or combined diet were subjected to ex vivo MRI, myographical, biomechanical, microscopical and histological analysis. Lipoproteins were analyzed by FPLC.Findings: We developed and applied an automated system for vessel wall injury that leads to more homogenous damage and more pronounced atherosclerotic plaque development even at low balloon pressure. Already in the absence of hypercholesterolemia, B vitamin deficiency results in accumulation of macrophages and lipids in the aorta, impairment of its biomechanical properties and disorganization of aortic collagen. Combination of B vitamin deficiency and hypercholesterolemia leads to thickening of the aorta, decreased aortic water diffusion, increased LDL-cholesterol and impaired vascular reactivity of the aorta.Interpretation: Our findings suggest that deficiency of B vitamins leads to atherosclerotic transformation of the aorta already in the absence of hypercholesterolemia and aggravates atherosclerosis development in its presence.Funding: This work was funded by the Austrian Science Fund (FWF) (Projects P31105 and P33672), the EU project NanoAthero and BioTechMed-Graz.

Aims Lipotoxic cardiomyopathy in diabetic and obese patients typically encompasses increased cardiac fatty acid (FA) uptake eventually surpassing the mitochondrial oxidative capacity. Lowering FA utilization via inhibition of lipolysis represents a strategy to counteract the development of lipotoxic heart dysfunction. However, defective cardiac triacylglycerol (TAG) catabolism and FA oxidation in humans (and mice) carrying mutated ATGL alleles provokes lipotoxic heart dysfunction questioning a therapeutic approach to decrease cardiac lipolysis. Interestingly, decreased lipolysis via cardiac overexpression of Perilipin 5 (Plin5), a binding partner of ATGL, is compatible with normal heart function and lifespan despite massive cardiac lipid accumulation. Herein, we decipher mechanisms that protect Plin5 transgenic mice from the development of heart dysfunction. Methods and results We generated mice with cardiac-specific overexpression of Plin5 encoding a serine-155 to alanine exchange (Plin5-S155A) of the protein kinase A phosphorylation site, which has been suggested as a prerequisite to stimulate lipolysis and may play a crucial role in the preservation of heart function. Plin5-S155A mice showed a substantial increase in cardiac TAG and ceramide levels, which was comparable to mice overexpressing non-mutated Plin5. Lipid accumulation was compatible with normal heart function even under mild stress. Plin5-S155A mice showed reduced cardiac FA oxidation but normal ATP production and changes in the Plin5-S155A phosphoproteome compared to Plin5 transgenic mice. Interestingly, mitochondrial recruitment of dynamin-related protein 1 (Drp1) was markedly reduced in cardiac muscle of Plin5-S155A and Plin5 transgenic mice accompanied by decreased phosphorylation of mitochondrial fission factor, a mitochondrial receptor of Drp1. Conclusions This study suggests that low cardiac lipolysis is associated with reduced mitochondrial fission and may represent a strategy to combat the development of lipotoxic heart dysfunction.

Atherosclerosis is the leading cause of cardiovascular disease, which is responsible for the majority of deaths worldwide. Lack of reproducible and controlled induction of atherosclerotic lesions in preclinical animal models limits atherosclerosis research. Here, we developed the first automated system for vessel wall injury that leads to more homogenous damage and more pronounced atherosclerotic plaque development at low balloon pressure in a rabbit model of atherosclerosis, and used this system to investigate the risk factor interplay. Elevated plasma homocysteine is an independent risk factor for atherosclerosis and is strongly linked to cardiovascular mortality. Already in the absence of hypercholesterolemia, deficiency in vitamins and choline required for homocysteine metabolization results in atherogenic transformation of the aorta i.e. impairment of its biomechanical properties, alteration of collagen organization as well as macrophage and lipid accumulation, and aggravation of atherosclerosis including significantly altered water diffusion and impaired vascular reactivity in its presence.

BACKGROUND amp; AIMS: Steatohepatitis (SH) and SH-associated hepatocellular carcinoma (HCC) are of considerable clinical significance. SH is morphologically characterized by steatosis, liver cell ballooning, cytoplasmic aggregates termedMallory-Denk bodies (MDBs), inflammation, and fibrosis at late stage. Disturbance of the keratin cytoskeleton and aggregation of keratins (KRTs) are essential for MDB formation. METHODS: Weanalyzed livers of aged Krt18(-/-) mice that spontaneously developed in the majority of cases SH-associated HCC independent of sex. Interestingly, the hepatic lipid profile in Krt18(-/-) mice, which accumulate KRT8, closely resembles human SH lipid profiles and shows that the excess of KRT8 over KRT18 determines the likelihood to develop SH-associated HCC linked with enhanced lipogenesis. RESULTS: Our analysis of the genetic profile of Krt18(-/-) mice with 26 human hepatoma cell lines and with data sets of amp;gt;300 patients with HCC, where Krt18(-/-) gene signatures matched human HCC. Interestingly, a high KRT8/18 ratio is associated with an aggressive HCC phenotype. CONCLUSIONS: We can prove that intermediate filaments and their binding partners are tightly linked to hepatic lipid metabolism and to hepatocarcinogenesis. We suggest KRT8/18 ratio as a novel HCC biomarker for HCC. Funding Agencies|Kurt und Senta Herrmann Stiftung; Austrian Genome Programme GEN-AU; DFG [MA1316-15, MA1316-17, MA1316-19, MA1316-21, INST 268/230-1]; German Research Foundation [STR 1095/4-2]; Else Kroner Exzellenzstipendium; Innovative Medicines Initiative Joint Undertaking from the European Unions Seventh Framework Programme (FP7/2007-2013) [115234]; EFPIA companies

Lysosomal acid lipase (LAL) hydrolyses cholesteryl esters and triacylglycerols (TG) within lysosomes to mobilise NEFA and cholesterol. Since LAL-deficient (Lal -/- ) mice suffer from progressive loss of adipose tissue and severe accumulation of lipids in hepatic lysosomes, we hypothesised that LAL deficiency triggers alternative energy pathway(s). We studied metabolic adaptations in Lal -/- mice. Despite loss of adipose tissue, Lal -/- mice show enhanced glucose clearance during insulin and glucose tolerance tests and have increased uptake of [3H]2-deoxy-D-glucose into skeletal muscle compared with wild-type mice. In agreement, fasted Lal -/- mice exhibit reduced glucose and glycogen levels in skeletal muscle. We observed 84% decreased plasma leptin levels and significantly reduced hepatic ATP, glucose, glycogen and glutamine concentrations in fed Lal -/- mice. Markedly reduced hepatic acyl-CoA concentrations decrease the expression of peroxisome proliferator-activated receptor α (PPARα) target genes. However, treatment of Lal -/- mice with the PPARα agonist fenofibrate further decreased plasma TG (and hepatic glucose and glycogen) concentrations in Lal -/- mice. Depletion of hepatic nuclear factor 4α and forkhead box protein a2 in fasted Lal -/- mice might be responsible for reduced expression of microsomal TG transfer protein, defective VLDL synthesis and drastically reduced plasma TG levels. Our findings indicate that neither activation nor inactivation of PPARα per se but rather the availability of hepatic acyl-CoA concentrations regulates VLDL synthesis and subsequent metabolic adaptations in Lal -/- mice. We conclude that decreased plasma VLDL production enhances glucose uptake into skeletal muscle to compensate for the lack of energy supply.

Clustering of biocompatible magnetic iron oxide nanocrystallites (MIONs) is a synthetic strategy which improves magnetic manipulation, imaging, and sensing for biomedical applications. In this work we describe the synthesis of condensed clustered MIONs obtained through biomineralization and epitaxial aggregation in the presence of alginate at ambient conditions, mimicking the process that so far has been achieved only by nature, in iron-oxidizing bacteria. These condensed-type magnetic nanostructures exhibit higher magnetophoretic responses compared to other types of magnetic colloids and clustered systems. The soft environmental conditions used for the synthesis of the magnetic nanosystems enables the alginate coating material to retain high drug loading ability for the doxorubicin molecule as well as strong binding proclivity for radionuclides. The strong binding of doxorubicin forms the physical basis to obtain magnetic nanocarriers, where the selective release of the drug occurs only under the action ...

Monoglyceride lipase (MGL) hydrolyzes monoglycerides (MGs) to glycerol and fatty acids. Among various MG species MGL also degrades 2-arachidonoylglycerol (2-AG), the most abundant endocannabinoid and potent activator of cannabinoid receptors (CBR) 1 and 2. MGL-knockout (−/−) mice exhibit pronounced 2-AG accumulation, but lack central cannabimimetic effects due to CB1R desensitization. We have previously shown that MGL affects plaque stability in apolipoprotein E (ApoE)−/− mice, an established animal model for dyslipidemia and atherosclerosis. In the current study, we investigated functional consequences of MGL deficiency on lipid and energy metabolism in ApoE/MGL double knockout (DKO) mice. MGL deficiency affected hepatic cholesterol metabolism by causing increased cholesterol elimination via the biliary pathway. Moreover, DKO mice exhibit lipid-triggered delay in gastric emptying without major effects on overall triglyceride and cholesterol absorption. The observed phenotype of DKO mice is likely not a consequence of potentiated CB1R signaling but rather dependent on the activation of alternative signaling pathways. We conclude that MGL deficiency causes complex metabolic changes including cholesterol metabolism and regulation of gut transit independent of the endocannabinoid system.

Quantitative magnetic resonance imaging (qMRI) offers several advantages in imaging and determination of soft tissue alterations when compared to qualitative imaging techniques. Although applications in brain and muscle tissues are well studied, its suitability to quantify relaxation times of intact and injured bone tissue, especially in children, is widely unknown. The objective observation of a fracture including its age determination can become of legal interest in cases of child abuse or maltreatment. Therefore, the aim of this study is the determination of time dependent changes in intact and corresponding injured bones in immature rats via qMRI, to provide the basis for an objective and radiation-free approach for fracture dating. Thirty-five MR scans of 7 Sprague-Dawley rats (male, 4 weeks old, 100 ± 5 g) were acquired on a 3T MRI scanner (TimTrio, Siemens AG, Erlangen, Germany) after the surgical infliction of an epiphyseal fracture in the tibia. The images were taken at days 1, 3, 7, 14, 28, 42 and 82 post-surgery. A proton density-weighted and a T1-weighted 3D FLASH sequence were acquired to calculate the longitudinal relaxation time T1 of the fractured region and the surrounding tissues. The calculation of T1 in intact and injured bone resulted in a quantitative observation of bone development in intact juvenile tibiae as well as the bone healing process in the injured tibiae. In both areas, T1 decreased over time. To evaluate the differences in T1 behaviour between the intact and injured bone, the relative T1 values (bone-fracture) were calculated, showing clear detectable alterations of T1 after fracture occurrence. These results indicate that qMRI has a high potential not only for clinically relevant applications to detect growth defects or developmental alterations in juvenile bones, but also for forensically relevant applications such as the dating of fractures in cases of child abuse or maltreatment.

Daniela Frascione,1 Clemens Diwoky,2 Gunter Almer,1,3 Peter Opriessnig,2 Caroline Vonach,1 Kerstin Gradauer,1 Gerd Leitinger,4 Harald Mangge,3 Rudolf Stollberger,2 Ruth Prassl1,51Institute of Biophysics and Nanosystems Research, Austrian Academy of Sciences, Graz, Austria; 2Institute of Medical Engineering, University of Technology, Graz, Austria; 3Clinical Institute for Medical and Chemical Laboratory Diagnosis (CIMCL), Medical University, Graz, Austria; 4Institute of Cell Biology, Histology and Embryology, Medical University, Graz, Austria; 5Ludwig Boltzmann Institute for Lung Vascular Research, Graz, AustriaBackground: Magnetic liposomes (MLs) are phospholipid vesicles that encapsulate magnetic and/or paramagnetic nanoparticles. They are applied as contrast agents for magnetic resonance imaging (MRI). MLs have an advantage over free magnetic nanocores, in that various functional groups can be attached to the surface of liposomes for ligand-specific targeting. We have synthesized PEG-coated sterically-stabilized magnetic liposomes (sMLs) containing ultrasmall superparamagnetic iron oxides (USPIOs) with the aim of generating stable liposomal carriers equipped with a high payload of USPIOs for enhanced MRI contrast.Methods: Regarding iron oxide nanoparticles, we have applied two different commercially available surface-coated USPIOs; sMLs synthesized and loaded with USPIOs were compared in terms of magnetization and colloidal stability. The average diameter size, morphology, phospholipid membrane fluidity, and the iron content of the sMLs were determined by dynamic light scattering (DLS), transmission electron microscopy (TEM), fluorescence polarization, and absorption spectroscopy, respectively. A colorimetric assay using potassium thiocyanate (KSCN) was performed to evaluate the encapsulation efficiency (EE%) to express the amount of iron enclosed into a liposome. Subsequently, MRI measurements were carried out in vitro in agarose gel phantoms to evaluate the signal enhancement on T1- and T2-weighted sequences of sMLs. To monitor the biodistribution and the clearance of the particles over time in vivo, sMLs were injected in wild type mice.Results: DLS revealed a mean particle diameter of sMLs in the range between 100 and 200 nm, as confirmed by TEM. An effective iron oxide loading was achieved just for one type of USPIO, with an EE% between 74% and 92%, depending on the initial Fe concentration (being higher for lower amounts of Fe). MRI measurements demonstrated the applicability of these nanostructures as MRI probes.Conclusion: Our results show that the development of sMLs is strictly dependent on the physicochemical characteristics of the nanocores. Once established, sMLs can be further modified to enable noninvasive targeted molecular imaging.Keywords: magnetic liposomes, fluorescence polarization, biodistribution, MRI contrast agent&nbsp

Summary Fatty acids (FAs) activate and fuel UCP1-mediated non-shivering thermogenesis (NST) in brown adipose tissue (BAT). Release of FAs from intracellular fat stores by adipose triglyceride lipase (ATGL) is considered a key step in NST. Accordingly, the severe cold intolerance of global ATGL knockout (AKO) mice has been attributed to defective BAT lipolysis. Here we show that this conclusion is incorrect. We demonstrate that although the BAT-specific loss of ATGL impairs BAT lipolysis and alters BAT morphology, it does not compromise the β3-adrenergic thermogenic response or cold-induced NST. Instead, NST depends on nutrient supply or lipolysis in white adipose tissue during fasting, suggesting that circulating energy substrates are sufficient to fuel NST. Cold intolerance in AKO mice is not caused by BAT dysfunction as previously suspected but by severe cardiomyopathy. We conclude that functional NST requires adequate substrate supply and cardiac function, but does not depend on ATGL-mediated lipolysis in BAT., Graphical Abstract, Highlights • ATGL in BAT is not a prerequisite for cold-induced thermogenesis in vivo • ATGL in WAT is crucial to liberate fatty acids for energy combustion during fasting • Loss of ATGL in the heart induces hypothermia due to insufficient cardiac reserve • Adaptive thermogenesis is normal in mice lacking ATGL in BAT, Schreiber et al. demonstrate that ATGL-mediated lipolysis in brown adipose tissue is not a prerequisite for cold-induced non-shivering thermogenesis in vivo. Instead, ATGL in white adipose tissue and the heart is essential for supplying fatty acids during fasting and for meeting increased demands on the cardiovascular system in the cold, respectively.

This article describes a new acquisition and reconstruction concept for positive contrast imaging of cells labeled with superparamagnetic iron oxides (SPIOs). Overcoming the limitations of a negative contrast representation as gained with gradient echo and fully balanced steady state (bSSFP), the proposed method delivers a spatially localized contrast with high cellular sensitivity not accomplished by other positive contrast methods. Employing a 3D radial bSSFP pulse sequence with half-echo sampling, positive cellular contrast is gained by adding artificial global frequency offsets to each half-echo before image reconstruction. The new contrast regime is highlighted with numerical intravoxel simulations including the point-spread function for 3D half-echo acquisitions. Furthermore, the new method is validated on the basis of in vitro cell phantom measurements on a clinical MRI platform, where the measured contrast-to-noise ratio (CNR) of the new approach exceeds even the negative contrast of bSSFP. Finally, an in vivo proof of principle study based on a mouse model with a clear depiction of labeled cells within a subcutaneous cell islet containing a cell density as low as 7 cells/mm3 is presented. The resultant isotropic images show robustness to motion and a high CNR, in addition to an enhanced specificity due to the positive contrast of SPIO-labeled cells. Copyright © 2014 John Wiley & Sons, Ltd.

Continuous monitoring of the oxygen saturation and the heart rate of sedated laboratory mice is achieved by adapting the principles of a human pulse oximeter. Using a measuring hardware with the proper analysis software in a micro-controller it is possible to calculate and display those vital signs periodically on a LCD and supply the digital data via a USB connection to a MATLAB GUI for further investigations. The results show an overall agreement with the theory and allow to use this work in preclinical studies to control the anaesthesia gases and the ventilation of sedated mice during research processes.

Core–shell hydrophilic superparamagnetic iron oxide (SPIO) nanoparticles, surface functionalized with either terephthalic acid or 2-amino terephthalic acid, showed large negative MRI contrast ability, validating the advantage of using low molecular weight and π-conjugated canopies for engineering functional nanostructures with superior performances.

Cachexia is a multifactorial wasting syndrome most common in patients with cancer that is characterized by the uncontrolled loss of adipose and muscle mass. We show that the inhibition of lipolysis through genetic ablation of adipose triglyceride lipase (Atgl) or hormone-sensitive lipase (Hsl) ameliorates certain features of cancer-associated cachexia (CAC). In wild-type C57BL/6 mice, the injection of Lewis lung carcinoma or B16 melanoma cells causes tumor growth, loss of white adipose tissue (WAT), and a marked reduction of gastrocnemius muscle. In contrast, Atgl-deficient mice with tumors resisted increased WAT lipolysis, myocyte apoptosis, and proteasomal muscle degradation and maintained normal adipose and gastrocnemius muscle mass. Hsl-deficient mice with tumors were also protected although to a lesser degree. Thus, functional lipolysis is essential in the pathogenesis of CAC. Pharmacological inhibition of metabolic lipases may help prevent cachexia.

Variable density random sampling patterns have recently become increasingly popular for accelerated imaging strategies, as they lead to incoherent aliasing artifacts. However, the design of these sampling patterns is still an open problem. Current strategies use model assumptions like polynomials of different order to generate a probability density function that is then used to generate the sampling pattern. This approach relies on the optimization of design parameters which is very time consuming and therefore impractical for daily clinical use. This work presents a new approach that generates sampling patterns by making use of power spectra of existing reference data sets and hence requires neither parameter tuning nor an a priori mathematical model of the density of sampling points. The approach is validated with downsampling experiments, as well as with accelerated in vivo measurements. The proposed approach is compared with established sampling patterns, and the generalization potential is tested by using a range of reference images. Quantitative evaluation is performed for the downsampling experiments using RMS differences to the original, fully sampled data set. Our results demonstrate that the image quality of the method presented in this paper is comparable to that of an established model-based strategy when optimization of the model parameter is carried out and yields superior results to non-optimized model parameters. However, no random sampling pattern showed superior performance when compared to conventional Cartesian subsampling for the considered reconstruction strategy.

Transcriptional regulation of small intestinal gene expression controls plasma total cholesterol (TC) and triglyceride (TG) levels, which are major determinants of metabolic diseases. GATA4, a zinc finger domain transcription factor, is critical for jejunal identity, and intestinal GATA4 deficiency leads to a jejunoileal transition. Although intestinal GATA4 ablation is known to misregulate jejunal gene expression, its pathophysiological impact on various components of metabolic syndrome remains unknown. Here, we used intestine-specific GATA4 knockout (GATA4iKO) mice to dissect the contribution of GATA4 on obesity development. We challenged adult GATA4iKO mice and control littermates with a Western-type diet (WTD) for 20 wk. Our findings show that WTD-fed GATA4iKO mice are resistant to diet-induced obesity. Accordingly, plasma TG and TC levels are markedly decreased. Intestinal lipid absorption in GATA4iKO mice was strongly reduced, whereas luminal lipolysis was unaffected. GATA4iKO mice displayed a greater glucagon-like peptide-1 (GLP-1) release on normal chow and even after long-term challenge with WTD remained glucose sensitive. In summary, our findings show that the absence of intestinal GATA4 has a beneficial effect on decreasing intestinal lipid absorption causing resistance to hyperlipidemia and obesity. In addition, we show that increased GLP-1 release in GATA4iKO mice decreases the risk for development of insulin resistance.

Subsampling of radially encoded MRI acquisitions in combination with sparsity promoting methods opened a door to significantly increased imaging speed, which is crucial for many important clinical applications. In particular, it has been shown recently that total variation (TV) regularization efficiently reduces undersampling artifacts. The drawback of the method is the long reconstruction time which makes it impossible to use in daily clinical practice, especially if the TV optimization problem has to be solved repeatedly to select a proper regularization parameter.The goal of this work was to show that for the case of MR Angiography, TV filtering can be performed as a post-processing step, in contrast to the common approach of integrating TV penalties in the image reconstruction process. With this approach, it is possible to use TV algorithms with data fidelity terms in image space, which can be implemented very efficiently on graphic processing units (GPUs). The combination of a special radial sampling trajectory and a full 3D formulation of the TV minimization problem is crucial for the effectiveness of the artifact elimination process.The computation times of GPU-TV show that interactive elimination of undersampling artifacts is possible even for large volume data sets, in particular allowing the interactive determination of the regularization parameter. Results from phantom measurements and in vivo angiography data sets show that 3D TV, together with the proposed sampling trajectory, leads to pronounced improvements in image quality. However, while artifact removal was very efficient for angiography data sets in this work, it cannot be expected that the proposed method of TV post-processing will work for arbitrary types of scans.

Abstract 699 Rationale: Vascular repair after hypoxic tissue damage requires a stringent interaction between somatic endothelial colony-forming progenitor cells (ECFCs) and mesenchymal stem and progenitor cells (MSPCs). Stem cell therapy to re-vascularize ischemic tissue has been a promising tool for various therapeutic targets including stroke, myocardial infarction and peripheral artery disease. Despite promising experimental data, therapeutic approaches employing endothelial progenitor cells have been of rather limited efficiency in clinical trials for both therapeutic vasculogenesis as well as anti-angiogenic therapy. Hypoxia in ischemic tissue is an extensively studied key factor that influences pro- and anti-angiogenic treatment by driving the revascularization machinery. We and others have shown that despite hypoxic stimulation, ECFCs in vivo only form patent vessels in the presence of MSPCs. Here we show that MSPCs but not ECFCs are the oxygen sensors enabling vasculogenesis in vivo. Methods: Adult human ECFCs were isolated from blood and MSPCs from bone marrow aspirates and expanded under humanized culture conditions. In in vitro studies progenitor cell phenotype, long-term proliferation, molecular cellular response, wound repair as well as migratory and vasculogenic functions were monitored under severe hypoxia (1% O2), venous oxygen conditions (5% O2) and standard culture conditions (20% O2). ECFC and MSPC interaction in vivo were studied in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) after subcutaneous transplantation in various extracellular matrices (matrigel, collagen/fibronectin, human platelete lysate). To investigate the respective roles of MSPCs and ECFCs during vasculogenesis under hypoxia in vivo chemical and genetic inhibitors against protein synthesis (cycloheximide) and HIF-1α (YC-1, shRNA) were employed. Immune histochemistry, immune fluorescence and TUNEL assays were performed on plugs in the time course after transplantation. Results: In vitro studies showed that compared to 20% O2, proliferation of ECFCs and MSPCs in primary and long-term cultures was significantly reduced at 5% O2, and even more at 1% O2. Standard culture conditions resulted in a shift in the progenitor hierarchy with an augmented number of high proliferative potential (HPP)-ECFC colonies (60±18% of total colonies) as compared to venous oxygen conditions (9±6%) and a complete loss of HPP-ECFC colonies under severe hypoxia (0%). The absolute colony number remained unchanged independent of oxygen levels. Both ECFC vascular wound repair function in scratch assays and the ability to form vascular-like networks in matrigel assays in vitro were diminished with declining oxygen supply. The re-oxygenation to 20% O2 of ECFCs which where precultured at 1% or 5% O2 led to enhanced proliferation, colony size and function. Single cell analysis revealed that ECFCs stabilized hypoxia-inducing factor-1α (HIF-1α) only at 1% O2 while MSPCs stabilize HIF-1α at 1% O2 as well as 5% O2 conditions. In a mouse model, subcutaneously injected ECFCs underwent apoptosis after 24h and attracted mouse leucocytes. In contrast, ECFCs co-implanted in vivo with MSPCs were rescued from apoptotic death and formed perfused human vessels 7 days after transplantation independent of matrix. Perivascular cells, but not ECFCs, were positive for HIF-1α in vivo. Inhibition of MSPCs but not ECFCs protein synthesis and HIF-1α prior to co-implantation blocked vessel formation. Conclusion: These data demonstrates that hypoxic ECFCs alone show reduced functuionality in vitro and form patent vessels in vivo. In contrast, MSPCs react to the low oxygen environment more sensitively than ECFCs and promote vessel formation at least in part by rescuing ECFCs from hypoxia-induced apoptosis. Surprisingly, this study shows that therapeutic vasculogenesis can occur independent of endothelial HIF stabilization and protein synthesis. This data indicate that in addition to their established role regulating hematopoiesis, MSPCs oxygen sensing is crucial during vascular regeneration. This suggests a shift of focus from endothelial cells to perivascular cells as a therapeutic target in regenerative medicine and anti-angiogenic therapy. Disclosures: No relevant conflicts of interest to declare.

Magnetoliposomes (MLs) are phospholipid vesicles encapsulating magnetic nanoparticles utilised as contrast agents for targeted molecular Magnetic Resonance Imaging (MRI). [...]

Objectives Bone bridge formation occurs after physeal lesions and can lead to growth arrest if not reversed. Previous investigations on the underlying mechanisms of this formation used histological methods. Therefore, this study aimed to apply a minimally invasive method using dynamic contrast-enhanced MRI (DCE-MRI). Materials and methods Changes in functional parameters related to the microvessel system were assessed in a longitudinal study of a cohort of an animal model applying a reference region model. The development of morphology of the injured physis was investigated with 3D high-resolution MRI. To acquire complementary information for MRI-related findings qRT-PCR and immunohistochemical data were acquired for a second cohort of the animal model. Results The evaluation of the pharmacokinetic parameters showed a first rise of the transfer coefficient 7 days post-lesion and a maximum 42 days after operation. The analysis of the complementary data showed a connection of the first rise to microvessel proliferation while the maximum value was linked to bone remodeling. Conclusion The pharmacokinetic analysis of DCE-MRI provides information on a proliferation of microvessels during the healing process as a sign for bone bridge formation. Thereby, DCE-MRI could identify details, which up to now required analyses of highly invasive methods.